A focus in our lab has been the study of mast cell-related diseases, including allergic diseases and mast cell (MC) proliferative disorders. The mediators (derived or not from MCs) and receptors that critically modulate or contribute to the pathological aspects of these disorders are, however, incompletely understood. Sphingosine-1-phosphate (S1P) signaling was identified as a canonical pathway downregulated in a group of patients with food anaphylaxis to lipid transfer protein (LTP), a protein present in nuts, vegetables, and fruits. Since S1P is an important lipid mediator for immunity, sensitization to food allergens and recovery from anaphylactic shock, we are investigating the role of S1P and its receptors in MCs and basophils, and in models of allergic inflammation. In FY2017 through FY2018, we identified the expression of the S1P4 receptor (S1pr4) in MCs and found that genetic deletion of S1pr4 resulted in exacerbation of passive systemic anaphylaxis to IgE/anti-IgE in mice. This phenotype was likely related to MC-extrinsic influences, such as the high circulating levels of IgE in these mice, which increases Fc-epsilon-RI expression and consequently the extent of the response to Fc-epsilon-RI engagement. Although S1P4 was dispensable for Fc-epsilon-RI induced MC responses under standard culture conditions, in the context of IL-33 co-stimulation, IgE-mediated degranulation was negatively modulated by S1P4, a finding of relevance, given the involvement of the IL-33-MC axis in allergic inflammation and food allergy. Further studies are needed to confirm a role for this receptor in the modulation of IL-33 mediated allergic inflammation. In FY2018, we have continued to study the effects of this receptor on other immune cells that may predispose to an allergy phenotype. Neoplastic accumulation of MCs in systemic mastocytosis (SM), a MC proliferation disorder, associates with activating mutations in the receptor tyrosine kinase KIT. Advanced SM can result in aggressive neoplastic MC infiltrates in tissues and end-of-organ compromise, requiring cytoreductive therapy. Current treatments targeting oncogenic D816V-KIT have shown limited long-term improvement and thus additional therapeutic approaches are needed. In FY2017 through FY2018, we found that SPHK1 and SPHK2, the enzymes responsible for the generation of the lipid mediator S1P, are upregulated in neoplastic human MCs and critically regulate their proliferation/survival. We also demonstrated the effectiveness of blockage of SPHK1 and SPHK2 in reducing the numbers of malignant bone marrow MCs from patients with SM and in vivo in xenograft models. Of particular interest, inhibition of SPHK1 prevented entry into mitosis and induced cell death most prominently in MCs with D816V-KIT. The data implicates the SPHK1/S1P axis as a critical pathway in oncogenic KIT signaling, leading to abnormal MC growth, and suggests the potential for SPHK inhibitors as alternatives to tyrosine kinase inhibitors in the treatment of aggressive SM. Studies during FY2018 have indicated that serum of patients with SM contains elevated concentrations of extracellular vesicles (EVs) with a MC signature, and which associate with disease severity. EVs are released by all cell types but particularly from malignant cells. A body of literature indicates that the distinct traits of EVs provide a unique form of cell-to-cell communication by delivering their protected cargoes into bystander or distant cells, thus altering biological and pathogenic processes. The identification of EVs with a MC signature in mastocytosis has opened the possibility that their specific cargos can be shuttled into other cells, dysregulating their function and thus contributing to some the pathological aspects associated with mastocytosis.